Increased Level of Vascular Endothelial Growth Factors by 4-hexylresorcinol is Mediated by Transforming Growth Factor-ß1 and Accelerates Capillary Regeneration in the Burns in Diabetic Animals.

4-Hexyl resorcinol (4HR) is an organic compound and has been used in skin care application. 4HR is an M2-type macrophage activator and elevates vascular endothelial growth factor (VEGF) expression via the hypoxia-inducible factor (HIF)-independent pathway. As endothelial cells are important in wound healing, the human umbilical vein endothelial cells (HUVECs) were treated with 4HR, and changes in VEGF-A, -C, and transforming growth factor-ß1 (TGF-ß1) expression were investigated. The administration of 4HR increased the expression level of VEGF-A, -C, and TGF-ß1. The application of TGF-ß1 protein also increased the expression level of VEGF-A and -C. Knockdown with small interfering RNA (siRNA) targeting to TGF-ß1 and the selective chemical inhibition (A83-01) to ALK5 confirmed the involvement of the TGF-ß signaling pathway in the 4-HR-mediated VEGFs expression. 4HR application in a burn model of diabetic rats demonstrated an increased level of angiogenic proteins with wound healing. Compared to sericin application, the 4HR application group showed more prominent capillary regeneration. Collectively, 4HR activated TGF-ß1/ALK5/VEGFs signaling in endothelial cells and induced vascular regeneration and remodeling for wound healing.

Retrospective comparative clinical study for silk mat application into extraction socket.

BACKGROUND: Silk mats have been approved for clinical trials by the Korean Food and Drug Administration as membranes for guided tissue regeneration (GTR). In this study, silk mat application was compared to high-density polytetrafluoroethylene (dPTFE) membrane application or no membrane group. METHODS: To compare the silk mat group to the dPTFE group or the no membrane group, a retrospective sample collection was conducted. Bony defects were measured at the time of extraction (T0) and then at 3 months (T1) and 6 months after extraction (T2) on a digital panoramic view. Bone gain (BG) was calculated by subtracting from the bony defect at T0 to the bony defect at each follow-up. RESULTS: The BG at T2 was 2.44 ± 2.49 mm, 4.18 ± 1.80 mm, and 4.24 ± 2.05 mm in the no membrane group, silk mat group, and dPTFE group, respectively. Both membrane groups had significantly higher BG than BG in the no membrane group at T2 (P < 0.05). CONCLUSIONS: Both membrane groups showed higher BG than the no membrane group.

4-Hexylresorcinol and silk sericin increase the expression of vascular endothelial growth factor via different pathways.

Angiogenesis plays an important role in active inflammation and wound healing. Our results showed that silk sericin and 4-hexylresorcinol (4HR) increased vascular endothelial growth factor (VEGF) expression in a dose-dependent manner in RAW264.7 cells. Unlike 4HR, silk sericin increased the expression of hypoxia inducible factor-1α (HIF-1α) and HIF-2α. Pretreatment with an HIF inhibitor decreased the sericin-induced increase in VEGF expression. However, the HIF inhibitor did not affect the 4HR-induced increase in VEGF expression. An inhibitor of matrix metalloproteinase (MMP) declined the 4HR-induced increase in VEGF expression. Silk sericin increased production of reactive oxygen species (ROS), whereas 4HR decreased ROS. M1 markers were increased by silk sericin treatment, and M2 markers were increased by 4HR treatment. VEGF and angiogenin expression were higher in rats treated with a 4HR-incorporated silk mat than in rats treated with a silk mat alone. In conclusion, silk sericin and 4HR increased VEGF expression in RAW264.7 cells via HIF-mediated and MMP-mediated pathways, respectively. Silk sericin exerted like pro-oxidant effects and 4HR exerted anti-oxidant effects. Rats treated with a 4HR-incorporated silk mat showed higher levels of VEGF and angiogenin than those treated with a silk mat alone.

The effects of proteins released from silk mat layers on macrophages.

BACKGROUND: The objective of this study was to evaluate the changes in gene expression after incubation of cells with proteins released from different silk mat layers. METHODS: A silk cocoon from Bombyx mori was separated into four layers of equal thickness. The layers were numbered from 1 to 4 (from the inner to the outer layer). The proteins were released by sonication of a silk mat layer in normal saline. The concentration of proteins was determined by spectrophotometry. They were incubated with RAW264.7 cells, and changes in the expression of genes were evaluated by cDNA microarray analysis and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: Layer 1 and 4 groups had higher protein concentrations compared to those in layer 2 and 3 groups. The genes associated with inflammation and angiogenesis showed significantly higher expression in layer 1 and 4 groups. The results of qRT-PCR were in agreement with those of the cDNA microarray analysis. CONCLUSIONS: The silk mat from the middle portion of the silkworm cocoon yielded a lower protein release and caused an insignificant change in the expression of genes that are associated with inflammation and angiogenesis.

Mandibular Reconstruction Using a Customized Three-Dimensional Titanium Implant Applied on the Lingual Surface of the Mandible.

A patient had a right mandibular defect due to resection of an ameloblastoma. Previously, the defect had been reconstructed by an iliac bone graft, and subsequently, a titanium mesh with xenograft was used. However, it was not successfully reconstructed. For the recovery of mandible continuity and rehabilitation of jaw movement, we manufactured a customized 3-dimensional titanium implant by computer-aided design and manufacturing and electron beam melting technology. This implant was designed to have a porous body structure and lingual plate. The customized implant was accurately inserted in the bony defect. As a result, the patient showed a normal range of mouth opening and jaw movement. New bone migration was observed in the porous structure of the implant. Although there was a slight plate exposure and lack of alveolar bone formation, the customized 3D titanium implant successfully reconstructed the mandibular discontinuous defect and recovered jaw movement.

Bone regeneration is associated with the concentration of tumour necrosis factor-α induced by sericin released from a silk mat.

To understand the osteogenic effect of the middle layer of the silk cocoon, sericin was examined for its cellular effects associated with tumor necrosis factor-α (TNF-α) signaling in this study. The fragmented sericin proteins in the silk mat were evaluated for the TNF-α expression level in murine macrophages. The concentration of protein released from silk mats was higher in the outermost and the innermost layers than in the middle layers, and the protein released from the silk mat was identified as sericin. The level of TNF-α in murine macrophages was dependent on the applied concentration of sericin, and the expression of genes associated with osteogenesis in osteoblast-like cells was dependent on the applied concentration of TNF-α. In animal experiments, silk mats from the middle layers led to a higher regenerated bone volume than silk mats from the innermost layer or the outermost layer. If TNF-α protein was incorporated into the silk mats from the middle layers, bone regeneration was suppressed compared with unloaded silk mats from the middle layers. Accordingly, silk mats from the silk cocoon can be considered to be a fragmented sericin-secreting carrier, and the level of sericin secretion is associated with TNF-α induction and bone regeneration.

Botulinum Toxin Conjugated With Silk Fibroin and 4-Hexylresorcinol.

PURPOSE: The objective of this study was to evaluate whether silk fibroin (SF) incorporated into 4-hexylresorcinol (4HR) could increase botulinum toxin-A (BTX-A) activity. MATERIAL AND METHODS: In total, 30 rats were used for this study. The animals were divided into 6 groups according to the injected materials (SA: saline only; SF; 4HR; B2: 2 units of BTX-A; B2 + SF + 4HR: combination of B2, SF, and 4HR; B5: 5 units of BTX-A). Serial sonography was used for the evaluation of muscle thickness after injection. Immunohistochemical staining was used for the evaluation of myosin type II (myo2) and Bcl-2 protein expression. RESULTS: The relative thickness of the masseter muscle in B2 group was 66.14% ±â€Š4.55% to the preinjection level; in B2 + SF + 4HR group was 54.59% ±â€Š4.83%, and in B5 group was 56.19% ±â€Š8.28%. Any BTX-injected group showed significantly lower value of the relative muscle thickness compared to SA, SF, or 4HR group (P < 0.001 for all). The difference of relative muscle thickness between B2 group and B2 + SF + 4HR group was statistically significant (P < 0.001). The intensity of myo2 immunostaining in B5, B2, and B2 + SF + 4HR group was significantly higher than those in the other groups (P < 0.05). CONCLUSIONS: When 2 units of BTX was incorporated to SF and 4HR, combination formula showed similar activity to those of 5 units of BTX.

Silk Fibroin-Alginate-Hydroxyapatite Composite Particles in Bone Tissue Engineering Applications In Vivo.

The aim of this study was to evaluate the in vivo bone regeneration capability of alginate (AL), AL/hydroxyapatite (HA), and AL/HA/silk fibroin (SF) composites. Forty Sprague Dawley rats were used for the animal experiments. Central calvarial bone (diameter: 8.0 mm) defects were grafted with AL, AL/HA, or AL/HA/SF. New bone formation was evaluated by histomorphometric analysis. To demonstrate the immunocompatibility of each group, the level of tumor necrosis factor (TNF)-α expression was studied by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) at eight weeks post implantation. Additionally, osteogenic markers, such as fibroblast growth factor (FGF)-23, osteoprotegerin (OPG), and Runt-related transcription factor (Runx2) were evaluated by qPCR or IHC at eight weeks post implantation. The AL/HA/SF group showed significantly higher new bone formation than did the control group (p = 0.044) and the AL group (p = 0.035) at four weeks post implantation. Additionally, the AL/HA/SF group showed lower relative TNF-α mRNA levels and higher FGF-23 mRNA levels than the other groups did at eight weeks post implantation. IHC results demonstrated that the AL/HA/SF group had lower TNF-α expression and higher OPG and Runx2 expression at eight weeks post implantation. Additionally, no evidence of the inflammatory reaction or giant cell formation was observed around the residual graft material. We concluded that the AL/HA/SF composite could be effective as a scaffold for bone tissue engineering.

Accelerated biodegradation of silk sutures through matrix metalloproteinase activation by incorporating 4-hexylresorcinol.

Silk suture material is primarily composed of silk fibroin and regarded as a non-resorbable material. It is slowly degraded by proteolysis when it is implanted into the body. 4-Hexylresorcinol (4HR) is a well-known antiseptic. In this study, the biodegradability of 4HR-incorporated silk sutures were compared to that of untreated silk sutures and polyglactin 910 sutures, a commercially available resorbable suture. 4HR-incorporated silk sutures exhibited anti-microbial properties. Matrix metalloproteinase (MMP) can digest a wide spectrum of proteins. 4HR increased MMP-2, -3, and -9 expression in RAW264.7 cells. MMP-2, -3, and -9 were able to digest not only silk fibroin but also silk sutures. Consequently, 59.5% of the 4HR-incorporated silk suture material remained at 11 weeks after grafting, which was similar to that of polyglactin 910 degradation (56.4% remained). The residual amount of bare silk suture material at 11 weeks after grafting was 91.5%. The expression levels of MMP-2, -3 and -9 were high in the 4HR-incorporated silk suture-implanted site 12 weeks after implantation. In conclusion, 4HR-treated silk sutures exhibited anti-microbial properties and a similar level of bio-degradation to polyglactin 910 sutures and induced higher expression of MMP-2, -3, and -9 in macrophages.

S100 and p65 expression are increased in the masseter muscle after botulinum toxin-A injection.

BACKGROUND: The purpose of this study was to compare the expression levels of p65 and S100 in the rat masseter muscle after the injection of different concentrations of botulinum toxin-A (BTX-A). METHODS: We injected either 5 or 10 U of BTX-A into both masseter muscle of rats. As a control group, the same volume of saline was injected. After 14 days, the animals were sacrificed. Subsequently, a biopsy and immunohistochemical staining of the samples were performed using a p65 or S100 antibody. RESULTS: The cross-sectional area of each myofibril was significantly reduced by BTX-A injection (P < 0.001). The expression of p65 and S100 increased significantly with increasing concentrations of BTX-A (P < 0.001). CONCLUSIONS: The injection of BTX-A into the masseter muscle induced muscle atrophy. Subsequently, p65 and S100 expression in myoblasts were increased for the protection of muscle cells.

Comparison of unprocessed silk cocoon and silk cocoon middle layer membranes for guided bone regeneration.

BACKGROUND: Silk cocoon is composed of multiple layers. The natural silk cocoon containing all layers was cut as a rectangular shape as defined as total group. The inner and outermost layers were removed from the total group and the remained mat was defined as the middle group. The objectives of this study was to compare the total group with the middle group as a barrier membrane for the guided bone regeneration. METHODS: The effects of these materials on the cellular proliferation and alkaline phosphatase (ALP) expression of MG63 cells were explored. For comparing bone regeneration ability, bilateral bone defects were created in calvarial areas in ten adult New Zealand white rabbits. The defects were covered with silk membranes of the middle group, with silk membrane of the total group used as the control on the contralateral side. The defects were allowed to heal for 4 and 8 weeks. Micro-computerized tomography (µCT) and histological examination were performed. RESULTS: The middle group exhibited a higher MTT value 48 and 72 h after treatment compared to the total group. ALP expression was also higher in the middle group. The results of µCT and histologic examination showed that new bone formation was significantly higher in the middle group compared to the total group 8 weeks postoperatively (P < 0.05). CONCLUSIONS: In conclusion, the middle layer of the silk cocoon supports guided bone regeneration better than unprocessed silk cocoon.

Powdered Wound Dressing Materials Made from wild Silkworm Antheraea pernyi Silk Fibroin on Full-skin Thickness Burn Wounds on Rats.

PURPOSE: This study evaluated powdered burn wound dressing materials from wild silkworm fibroin in an animal model. METHODS: Fifteen rats were used in this experiment. Full-thickness 2×2 cm burn wounds were created on the back of rats under anesthesia. In the two experimental groups, the wounds were treated with two different dressing materials made from silkworm fibroin. In the Control Group, natural healing without any dressing material was set as control. The wound surface area was measured at five days, seven days and 14 days. Wound healing was evaluated by histologic analysis. RESULTS: By gross observation, there were no infections or severe inflammations through 14 days post-injury. The differences among groups were statistically significant at seven days and 14 days, postoperatively (P <0.037 and 0.001, respectively). By post hoc test, the defect size was significantly smaller in experimental Group 1 compared with the Control Group and experimental Group 2 at seven days postoperatively (P =0.022 and 0.029, respectively). The difference between Group 1 and Group 2 was statistically significant at 14 days postoperatively (P <0.001). Group 1 and control also differed significantly (P =0.002). Group 1 showed a smaller residual scar than the Control Group and Group 2 at 14 days post-injury. Histologic analysis showed more re-epithelization in Groups 1 and 2 than in the Control Groups. CONCLUSION: Burn wound healing was accelerated with silk fibroin spun by wild silkworm Antheraea pernyi. There was no atypical inflammation with silk dressing materials. In conclusion, silk dressing materials can be used for treatment of burn wound.

Silk fibroin and 4-hexylresorcinol incorporation membrane for guided bone regeneration.

The objective of this study was to demonstrate that a silk fibroin (SF) and 4-hexylresorcinol (4-HR) incorporation membrane could be used for a guided bone regeneration technique. Fourier transform infrared measurements were obtained to determine change of physical property of SF membrane by 4-HR incorporation. Two peri-implant defects, 3.0 × 5.0 mm (width × length), were prepared on the lateral side of the implant hole in the tibia of New Zealand white rabbits (n = 8). The peri-implant defect was left unfilled in the control group. Silk fibroin + 4-HR membrane was applied to the peri-implant defect in the experimental group. The 8 animals were killed at 8 weeks after implantation. Subsequently, removal torque test and histomorphometric evaluation were done. Fourier transform infrared spectroscopy showed no specific chemical interaction between 4-HR and SF. In the histomorphometric analysis, the mean bone regeneration was 18.3 ± 1.9 mm(2) in the experimental group and 9.3 ± 0.9 mm(2) in the control group (P = 0.004). In conclusion, the SF and 4-HR incorporation membrane successfully regenerated bone in the rabbit tibia peri-implant bone defect model.

Silk fibroin hydrolysate inhibits osteoclastogenesis and induces apoptosis of osteoclasts derived from RAW 264.7 cells.

Bone disease can be associated with bone resorption by osteoclasts, and interest in the development of antiresorptive agents has recently increased. The hydrolysate of silk fibroin has been studied with respect to such biomedical applications. In a previous study, silk fibroin showed indirect inhibitory effects on the differentiation of osteoclasts. To further evaluate the effect of a hydrolysate of silk fibroin on osteoclasts, we investigated the direct effects of the silk fibroin hydrolysate on osteoclastogenesis and apoptosis of osteoclasts induced by receptor activation of nuclear factor κB ligand (RANKL). The silk fibroin hydrolysate inhibited RANKL-induced formation of tartrate-resistant acid phosphatase (TRAP) in RAW 264.7 cells. The inhibitory effect of the silk fibroin hydrolysate resulted in the decreased expression of osteoclast marker genes, such as matrix metalloproteinase-9 (MMP-9), cathepsin-K and calcitonin receptor (CTR). In addition, the silk fibroin hydrolysate blocked the signaling pathways of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) and expression of transcription factors, such as nuclear factor of activated T cells c1 (NFATc1) and NF-κB. Finally, the silk fibroin hydrolysate induced apoptosis signaling cascades. Taken together, the present results indicate that silk fibroin hydrolysate has antiresorptive activity by both inhibiting osteoclastogenesis and inducing osteoclast apoptosis.

Biodegradation behavior of silk fibroin membranes in repairing tympanic membrane perforations.

Silk fibroin (SF) from silkworms has been widely studied as a biomaterial. The degradation behavior of silk biomaterials is important for medical applications, but few studies have examined long-term degradation behavior in vivo. In this study, we investigated the degradation behavior of SF membranes in vitro and in vivo. For the in vitro assay, we observed degradation of silk membranes in phosphate buffered saline, culture media, and an enzyme (proteinase K) solution. In the proteinase K solution, 80% of the silk membranes degraded within 10 days. Silk membranes exhibited no cytotoxicity toward L929 cells and rat tissues. To investigate the degradation of silk membranes in vivo, they were implanted subcutaneously in rats and harvested 19 months after surgery. Scanning electron microscopy imaging and histological analysis of silk membrane explants showed that they broke into several pieces after 16 months. Results show that silk membranes are biocompatible and display excellent long-term degradation behavior when used as biomaterials.

Enhancement of anti-inflammatory activity of PEP-1-FK506 binding protein by silk fibroin peptide.

Silk fibroin (SF) peptide has been traditionally used as a treatment for flatulence, spasms, and phlegm. In this study, we examined whether SF peptide enhanced the antiinflammatory effect of PEP-1-FK506 binding protein (PEP-1-FK506BP) through comparing the anti-inflammatory activities of SF peptide and/or PEP-1-FK506BP. In the presence or absence of SF peptide, transduction levels of PEP-1-FK506BP into HaCaT cells and mice skin and anti-inflammatory activities of PEP-1-FK506BP were identified by Western blot and histological analyses. SF peptide alone effectively reduced both mice ear edema and the elevated levels of cyclooxygenase-2, interleukin-6 and -1beta, and tumor necrosis factor-alpha, showing similar anti-inflammatory effect to that of PEP-1-FK506BP. Furthermore, co-treatment with SF peptide and PEP-1- FK506BP exhibited more enhanced anti-inflammatory effects than the samples treated with SF peptides or PEP- 1-FK506BP alone, suggesting the possibility that SF peptide and PEP-1-FK506BP might interact with each other. Moreover, the transduction data demonstrated that SF peptide did not affect the transduction of PEP-1- FK506BP into HaCaT cells and mice skin, indicating that the improvement of anti-inflammatory effect of PEP-1- FK506BP was not caused by enhanced transduction of PEP-1-FK506BP. Thus, these results suggest the possibility that co-treatment with SF peptide and PEP-1-FK506BP may be exploited as a useful therapy for various inflammationrelated diseases.

Effect of silk fibroin peptide derived from silkworm Bombyx mori on the anti-inflammatory effect of Tat-SOD in a mice edema model.

We investigated whether silk fibroin peptide derived from the silkworm, Bombyx mori, could inhibit inflammation and enhance the anti-inflammatory activity of Tat-superoxide dismutase (Tat-SOD), which was previously reported to effectively penetrate various cells and tissues and exert anti-oxidative activity in a mouse model of inflammation. Inflammation was induced by topical treatment of mouse ears with 12-O-tetradecanoylphorbol-13-acetate (TPA). Histological, Western blot, and reverse transcription-polymerase chain reaction data demonstrated that silk fibroin peptide or Tat-SOD alone could suppress elevated levels of cyclooxygenase-2, interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha induced by TPA. Moreover, silk fibroin peptide significantly enhanced the anti-inflammatory activity of Tat-SOD, although it had no influence on in vitro and in vivo transduction of Tat-SOD. Silk fibroin peptide exhibited anti- inflammatory activity in a mice model of inflammation. Therefore, silk fibroin peptide alone or in combination with Tat-SOD might be used as a therapeutic agent for various inflammatory diseases.

Accelerated healing with the use of a silk fibroin membrane for the guided bone regeneration technique.

OBJECTIVE: The purpose of this study was to evaluate the bone regeneration ability of silk fibroin (SF) membrane. STUDY DESIGN: Fourier-transform infrared (FT-IR) and solubility test against distilled water were performed with 3 different types of SF membrane (SM1, SM2, and SM3). Subsequently, microscopic computerized tomography (µ-CT) and histomorphometric analyses were performed in rabbit calvarial defect model after SF membrane application at 4 and 8 weeks after surgery. RESULTS: FT-IR showed that the conformation of the SF membrane was a random coil structure and that SM1 was the least soluble. When SM1 was used in the animal model, the groups with SM1 had significantly higher new bone formation than the uncovered control in both the µ-CT and the histomorphometric analyses (P < .05). CONCLUSIONS: The SF membrane had more new bone formation compared with the uncovered control.

Low molecular weight silk fibroin increases alkaline phosphatase and type I collagen expression in MG63 cells.

Silk fibroin, produced by the silkworm Bombyx mori, has been widely studied as a scaffold in tissue engineering. Although it has been shown to be slowly biodegradable, cellular responses to degraded silk fibroin fragments are largely unknown. In this study, silk fibroin was added to MG-63 cell cultures, and changes in gene expression in the MG-63 cells were screened by DNA microarray analysis. Genes showing a significant (2-fold) change were selected and their expression changes confirmed by quantitative RT-PCR and western blotting. DNA microarray results showed that alkaline phosphatase (ALP), collagen type-I alpha-1, fibronectin, and transforming growth factor-beta1 expressions significantly increased. The effect of degraded silk fibroin on osteoblastogenic gene expression was confirmed by observing up-regulation of ALP activity in MG-63 cells. The finding that small fragments of silk fibroin are able to increase the expression of osteoblastogenic genes suggests that controlled degradation of silk fibroin might accelerate new bone formation. [BMB reports 2010; 43(1): 52-56].

A combination graft of low-molecular-weight silk fibroin with Choukroun platelet-rich fibrin for rabbit calvarial defect.

OBJECTIVE: The objective of this study was to determine the capabilities of silk fibroin as a biomaterial template for bone formation when mixed with Choukroun platelet-rich fibrin (PRF) in vivo. STUDY DESIGN: Ten New Zealand white rabbits were used for this study and bilateral round shaped defects were formed in the parietal bone (diameter 9.0 mm). The silk fibroin was digested by acid and made into powder (molecular weight <1.0 kDa). The right side (experimental group) received the silk fibroin plus platelet-rich fibroin and the left side (control group) did not receive a graft. Animals were killed at 6 weeks and 12 weeks. The specimens were examined by microscopic computerized tomography (micro-CT). Subsequently, they underwent decalcification and were stained for histologic analysis. RESULTS: There was no significant difference between groups at 6 weeks after operation. In the micro-CT results, however, tissue mineral content in the experimental group at 12 weeks after operation was 132.09 +/- 4.41 and that in the control group was 126.42 +/- 6.62 (P = .011). Tissue mineral density in the experimental group was 2,088.88 +/- 648.34, and that in the control group was 2,029.72 +/- 668.22 (P = .013). The results of the histomorphometric analysis were in accordance with the micro-CT results. The total new bone was 49.86 +/- 7.49% in the control group at 12 weeks after the operation and 59.83 +/- 10.92% in the experimental group (P = .021). CONCLUSION: A combined application of Choukroun PRF with acid-digested silk fibroin showed more rapid bone healing than unfilled control.